1. Field of the Invention
This invention relates to a continuous culture of pluripotent parenchymal hepatocytes derived from the epiblast of pig blastocyst. The culture has been passaged continuously for over 2 years, indicating an infinitely self-renewing cell population. The stem cell characteristics of the hepatocytes indicate that the cells are unique for investigations of liver differentiation and organogenesis.
2. Description of the Prior Art
The continuous secondary culture of normal parenchymal hepatocytes has previously been unsuccessful (Williams et al., In Vitro, 13: 809-817, 1977; Guguen-Guillouzo et al., Exp. Cell Res., 143: 47-54, 1983; Perraud et al., Exp. Cell Res., 195: 59-65, 1991). In vitro hepatocyte studies have largely been limited to the use of primary cultures of hepatocytes established after collagenase perfusion of rat or mouse livers (Williams et al., 1977, supra; Berry and Friend, J. Cell Biol., 43: 506-520, 1969). These techniques were also amenable to the establishment of primary hepatocyte cultures in non-rodent species such as the pig (Caperna et al., J. Anim. Sci., 61: 1576-1586, 1985). However, the use of primary cultures is problematic since it is labor intensive and inherently variable. In addition, other epithelial cells, fibroblasts, and macrophages were common "contaminants" in primary liver cell culture and these cells can quickly overgrow or otherwise interfere not only with maintaining cultures but also with experimental manipulations and measurements (Caperna et al., 1985, supra; Furukawa et al., In Vitro, 23: 339-348, 1987; Langenbach et al., Cancer Res., 39: 3509-3514, 1979). Moreover, normal liver parenchymal cell activities such as .alpha.-fetoprotein (AFP) production, albumin production, and various enzymatic functions were often lost within one to two weeks of establishment in primary culture (Guguen-Guillouzo et al., 1983, supra; Langenbach et al., 1979, supra).
An alternative to normal parenchymal hepatocyte cell culture has been the establishment of numerous human and rodent hepatocarcinoma derived cell lines (Richardson et al., J. Cell Biol., 40: 236-247, 1969; Aden et al., Nature (London), 282: 615-616, 1970). Although several of these hepatoma cell lines were minimally deviated and expressed various proteins representative of normal hepatocyte function, they were likely to be abnormal in several aspects, particularly growth control. For example, some of these cell lines were highly tumorigenic when placed in vivo (Richardson, 1969, supra; Knowles et al., Science, 209: 497-499, 1980). Also, while hepatocyte growth factor (HGF) is mitogenic for primary hepatocytes, it has been reported to be cytostatic for the hepatoma cell lines HepG2, Hep3B, and H35 (Higashi and Shima, 1993). Some human hepatoma cell lines also chronically produced hepatitis proteins (Aden et al., 1979, supra; Knowles et al., 1980, supra). Thus, hepatoma derived cell cultures may confuse assessments of normal parenchymal hepatocyte biology in vitro, and in vivo assessments may not be possible. The ability to sustainably culture normal, functional parenchymal hepatocytes in secondary culture is therefore novel and of great value for the routine study of hepatocyte biology.
It has been hypothesized that the liver contains stem cells which support its general long term function, and its regenerative ability (Sigal et al., M. J. Physiol., 263: G139, 1992; Fausto and Meade, Lab. Invest., 60: 4-13, 1989). Stem cells have not been identified, although embryonic phenotypic gene expression, e.g., AFP, was found in some proliferating liver epithelia following the administration of hepatotoxic agents (Fausto et al., Purification and culture of oval cells from rat liver. In: Pretlow and Pretlow, eds. Cell Separation: Methods and Selected Applications, Vol. 4. Orlando: Academic Press, 45-77; Evarts et al., Carcinogenesis, 8: 1737-1740, 1987). Liver "oval" cells and intrahepatic bile duct cells were also proposed as candidate liver stem cells or facultative stem cells (Sigal et al., 1992, supra; Fausto and Meade, 1989, supra). A consensus of data indicated that liver stem cells would express AFP and albumin and would be capable of differentiating into bile duct cells and parenchymal hepatocytes (Shiojiri, N., J. Embryol. Exp. Morphol., 79: 149-152, 1981; Evarts et al. Cancer Res., 49: 1541-1547, 1989; Germain et al., Cancer Res., 48: 4909-4918, 1988).
Thus, although attempts have been made to establish normal hepatocyte cell lines which are capable of culturing over a long period of time (i.e., years) without losing characteristic activity, accomplishment of this goal has heretofore been unsuccessful.